Decapentaplegic-responsive silencers contain overlapping Mad-binding sites

Smad proteins regulate transcription in response to transforming growth factor-beta signaling pathways by binding to two distinct types of DNA sites. The sequence GTCT is recognized by all receptor-activated Smads and by Smad4. The subset of Smads that responds to bone morphogenetic protein signaling recognizes a distinct class of GC-rich sites in addition to GTCT. Recent work has shown that Drosophila Mad protein, the homologue of bone morphogenetic protein rSmads, binds to GRCGNC sites through the same MH1 domain beta-hairpin interface used to contact GTCT sites. However, binding to GRCGNC requires base-specific contact by two Mad proteins, and here we provide evidence that this is achieved by contact of the two Mad subunits that overlap across the two central base pairs of the site. This topology is supported by results indicating that His-93, which is located at the tip of the Mad beta-hairpin, is in close proximity to base pairs 2 and 5. Also consistent with the model is disruption of binding by mutation of Glu-39 and Glu-40, which are predicted to lie at the interface of the two overlapping Mad MH1 domains. As predicted from the overlapping model, binding is disrupted by insertion of 1 bp in the middle of the site, whereas insertion of 2 bp creates abutting sites that can be bound by the Mad-Medea heterotrimer without requiring Glu-39 and Glu-40. Overlapping Mad sites predominate in decapentaplegic response elements, consistent with a high degree of specificity in response to signaling.